ABSTRACT Cancer stem cells (CSCs) have been proposed recently to be the cause cancer initiation, progression and chemotherapy failure in pancreatic cancer. Abberant hedgehog signaling occurs in pancreatic cancer during the progression of PanIN lesions to metastatic tumors, suggesting that sonic hedgehog (Shh) signaling may be an early event leading to accumulation of undifferentiated precursor cells in pancreatic cancer. This project is based on the premise that sulforaphane (SFN), a natural compound from the cruciferous vegetables, can be used for prevention and/or treatment of human pancreatic cancer. Unfortunately, the intracellular mechanisms by which SFN inhibits growth and metastasis, and induces apoptosis in pancreatic cancer cells and CSCs have never been examined. Thus, the main objective of this application is to examine the molecular mechanisms by which sulforaphane inhibits pancreatic CSC characteristics, and assess the chemopreventive / therapeutic potential of sulforaphane against pancreatic cancer by targeting CSCs. These studies will empower us with new knowledge that will guide us in formulating strategies for prevention of human pancreatic cancer by targeting CSCs. We have recently demonstrated that human CD133+CD44+CD24+ESA+ CSCs express Nanog, Notch-1 and Oct-4 and are highly tumorigenic in mice. Our preliminary data indicate that: (i) SFN inhibits self-renewal capacity of pancreatic CSCs; (ii) SFN inhibits the expression of pluripotency maintaining factors (Nanog, Oct-4 and Sox-2), epithelial mesenchymal (EMT) markers (Zeb-1, Snail, Slug), Bcl-2 and XIAP in pancreatic CSCs; (iii) SFN inhibits the expression of Smo, Ptch as well as effector molecule Gli 1 and 2, suggesting the clinical significance of Shh pathway in pancreatic cancer; (iv) enforced activation of Shh pathway or inhibition of Gli1 plus Gli2 expression by shRNA blocked the inhibitory effects of SFN, suggesting these effects of sulforaphane are mediated through Shh pathway; and (v) SFN inhibits self-renewal capacity of CSCs (CD133+CD44+CD24+ESA+ cells with high Nanog) isolated from pancreas of human primary tumors and KrasG12D mice. Based on our preliminary findings, we hypothesize that sulforaphane alone or in combination with standard chemotherapeutic drugs inhibit pancreatic cancer stem cell characteristics by regulating pluripotency promoting factors and inhibiting the sonic hedgehog pathway. The above hypothesis will be tested by the following four specific aims: Aim 1. To examine the molecular mechanisms by which sulforaphane inhibits human pancreatic cancer stem cell characteristics and sensitizes drug- resistant cancer stem cells in vitro. Aim 2. To examine whether sulforaphane inhibits self-renewal capacity, migration, invasion and epithelial mesenchymal transition of pancreatic cancer stem cells through sonic hedgehog pathway and further to examine the involvement of Shh pathway in tumor-stroma interaction. Aim 3. To examine the effects of oral administration of sulforaphane on pancreatic carcinogenesis using a KrasG12D transgenic mouse model. Aim 4. To validate whether oral administration of sulforaphane is effective in inhibiting pancreatic cancer stem cell characteristics and tumor growth by inhibiting sonic hedgehog pathway, and to examine the interactive effects of sulforaphane with standard chemotherapeutic drugs in a NOD/SCID/IL2Rgammanull mouse model. Our studies are very novel and significant because they have prognostic relevance and inhibit pancreatic cancer initiation and progression by targeting CSCs.